Use of a nitroxide or a prodrug thereof in the prophylactic and therapeutic treatment of cancer

ABSTRACT

The present invention provides a method for the prophylactic and therapeutic treatment of cancer. The method comprises administering to an animal, preferably a mammal, more preferably a human, at risk for developing a cancer or having a cancer a nitroxide or a prodrug thereof, wherein the nitroxide or prodrug thereof preferably is alicyclic or heterocyclic and more preferably is a compound of Formula I or Formula II:  
                 
in an amount sufficient to prevent or treat said cancer, wherein said cancer is susceptible to prevention or treatment by said nitroxide or prodrug thereof. Also provided is a composition for use in the method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of copending U.S. patentapplication Ser. No. 09/424,519, which is the national phase ofPCT/US98/10685, filed May 27, 1998, claiming the benefit of U.S.Provisional Patent Application No. 60/047,724, filed May 27, 1997.

FIELD OF THE INVENTION

The present invention relates to nitroxides and prodrugs thereof andtheir use in the prophylactic and therapeutic treatment of cancer.

BACKGROUND OF THE INVENTION

Cancer is a major world-wide health problem. Given that the vastmajority of human tumors are difficult to treat effectively, thoseafflicted suffer physically, emotionally and financially and inevitablydie an early death. There is also a tremendous burden on the familiesand friends of those afflicted as well as on society at large.Accordingly, the ability to prevent cancer, delay its onset and/or slowits progression would benefit everyone.

Although extensive research around the world has led to advances incancer treatment, progress has been slow and there is no known cure.However, modern molecular biological techniques have contributed to ourunderstanding of the genetic aspects of cancer development. For example,the tumor suppressor gene p53, which is representative of a generalclass of genes that code for products that regulate cellular function bythwarting the cascade of events that causes a normally functioning cellto either die or become immortal, i.e., cancerous, has been shown toencode a transcription factor that suppresses tumor development.Mutations in the p53 tumor suppressor gene have been shown to affect theproduction of the oncogene-suppressing transcription factor. Forexample, either no transcription factor is produced or a transcriptionfactor that is ineffectual or partially effective is produced. In fact,the p53 tumor suppressor gene is the most common site of genetic lesionsin human cancers (Levine et al., Nature 351: 453-456 (1991); andHollstein et al., Science 253: 49-53 (1991)), with more than half of allhuman tumors exhibiting p53 point mutations or deletions (Chang et al.,Am. J. Gastroenterol. 88: 174-186 (1993)). Mutations in the p53 genealso have been associated with Li-Fraumeni syndrome, a familialautosomal dominant disease associated with an increased risk oftumorigenesis (Srivastava et al., Nature 348: 747-749 (1990)). The p53protein also plays a role in the cellular response to DNA-damagingagents by facilitating a block in the G1 phase of the cell cyclefollowing DNA damage, thereby providing time for repair of the DNAdamage (Pietenpol et al., Nature 365: 17-18 (1993); and Kuerbitz et al.,PNAS USA 89: 7491-7495 (1992)) or by causing apoptosis (Yonish-Rouach etal., Nature 352: 345-347 (1991)).

In order to enable the further study of the p53 gene, recombinant DNAtechniques have been used to develop rodent models. In one model, therodents are homozygous for mutant p53 alleles (p53−/−), such that thep53 gene is disrupted or “knocked-out” (p53−/−) and does not function,and the rodents are highly susceptible at an early age to a variety oftumors (Donehower et al., Nature 356: 251-221 (1992)). In another model,the rodents are heterozygous for wild-type and mutant p53 alleles(p53+/−) and, although they develop tumors 10-20 months after birth,they live considerably longer than the homozygous mutant p53 rodents(Harvey et al., Nature/Genetics 5: 225-229 (1993)). Exposure of theserodents to carcinogens, such as dimethylnitrosamine, or whole bodyirradiation accelerates tumor formation (Harvey et al. (1993), supra;and Lee et al., Oncogene 12: 3731-3736 (1994)).

Nitroxides are stable compounds, which are low in molecular weight,metal-independent, nontoxic and nonallergenic, and are characterized bylow reactivity with oxygen, high solubility in aqueous solutions, andthe ability to cross cellular membranes. The lipophilicity of nitroxidescan be controlled by the addition of various organic substituents, inorder to facilitate the targeting of the nitroxides to specific organsor organelles.

Nitroxides have been shown to protect cells and animals against theuntoward acute effects, such as cytotoxicity, of short-term exposure tolethal doses of free radicals and oxidative species, such as superoxide,hydrogen peroxide, hydroxyl radicals, and hydroperoxides, i.e., byfunctioning as antioxidants (U.S. Pat. No. 5,462,946). In cell culture,nitroxides have been shown to sensitize hypoxic cells to ionizingradiation and, paradoxically, protect aerobic cells from ionizingradiation. Also in cell culture, nitroxides have been shown to protectcells against the acute cytotoxic affects of paraquat andanti-neoplastic agents. In animals, nitroxides have been shown toprotect against radiation-induced alopecia and to induce weight loss. Ithas been reported that nitroxides can be used to protect againstpulmonary adult respiratory distress syndrome, lenticular degenerationand hyaline membrane disease in infants, cataracts, oxidative stress,such as that associated with oxygen therapy or hyperbaric oxygentreatment, reperfusion injury, such as that associated with myocardialinfarction, stroke, pancreatitis, intestinal ulceration, and organtransplantation.

It has now been surprisingly and unexpectedly discovered that nitroxidesand prodrugs thereof are useful in the prophylactic and therapeutictreatment of cancer (i.e., prevention, delay of onset, and slowing ofprogression of cancer). Accordingly, it is an object of the presentinvention to provide a method for the prophylactic and therapeutictreatment of cancer. It is another object of the present invention toprovide a composition for use in the method. These and other objects andadvantages of the present invention, as well as additional inventivefeatures, will be apparent from the description of the inventionprovided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method for the prophylactic andtherapeutic treatment of cancer. The method comprises administering toan animal, preferably a mammal, more preferably a human, at risk fordeveloping a cancer or having a cancer, a nitroxide or a prodrug thereofin an amount sufficient to prevent or treat the cancer, respectively,wherein said cancer is susceptible to prevention or treatment with saidnitroxide or said prodrug thereof. Preferably, the nitroxide or prodrugthereof is alicyclic or heterocyclic. More preferably, the nitroxide orprodrug thereof is a compound of Formula I or Formula II:

wherein R₁ is selected from the group consisting of H, OH, OZ, O., ═Oand Y, wherein Y is a leaving group, which can be converted to H, OH, O.or ═O by reaction with a nucleophilic agent, and Z is selected from thegroup consisting of a C₁₋₂₀ aliphatic group, a monocyclic aromaticgroup, a bicyclic aromatic group, a multicyclic aromatic group, a C₁₋₂₀alicyclic group, a noncarbon/nonoxygen moiety, a carbohydrate, a lipid,a nucleic acid and a protein. Preferably, the aromatic group comprises a5- or 6-membered structure in which each member is independentlyselected from the group consisting of carbon and a heteroatom. Preferredheteroatoms in the aromatic group include nitrogen, oxygen, sulfur,phosphorus and boron. The noncarbon/nonoxygen moiety preferablycomprises a member selected from the group consisting of boron, sulfur,phosphorus and nitrogen. R₂, R₃, R₄ and R₅ are independently selectedfrom the group consisting of a C₁₋₂₀ alkyl group, a C₂₋₂₀ alkenyl group,a C₂₋₂₀ alkynyl group, and —CH₂—[CR′R″]_(m)—CH₃, wherein R′ is selectedfrom the group consisting of hydrogen, a C₁₋₂₀ aliphatic group, amonocyclic aromatic group as described above, a bicyclic aromatic groupas described above, and a multicyclic aromatic group as described above,and R″ is selected from the group consisting of hydrogen, a C₁₋₂₀aliphatic group, a monocyclic aromatic group as described above, abicyclic aromatic group as described above, and a multicyclic aromaticgroup as described above, a C₁₋₂₀ alicyclic group, a noncarbon/nonoxygenmoiety as described above, a carbohydrate, a lipid, a nucleic acid, anda protein, and m≦30. R₂ and R₃ or R₄ and R₅ can be connected through oneor more members, each of which is independently selected from the groupconsisting of carbon and a heteroatom. R₆, R₇, R₈ and R₉ areindependently selected from the group consisting of hydrogen, a hydroxylgroup, a C₁₋₂₀ aldehydic group, a C₁₋₂₀ keto group, a primary aminogroup, a secondary amino group, a tertiary amino group, a sulfido group,a disulfido group, a sulfato group, a sulfito group, a sulfonato group,a sulfinato group, a sulfenato group, a sulfamato group, ametal-containing group, wherein the metal is preferably selected fromthe group consisting of a transition metal and a lanthanide, a siliconegroup, a halide, a C₁₋₂₀ ester-containing group, a carboxyl group, aphosphato group, a phosphino group, a phosphinato group, a phosphonatogroup, a C₁₋₂₀ alkyl group, a C₂₋₂₀ alkenyl group, a C₂₋₂₀ alkynylgroup, and —CH₂—[CR′R″]_(m)—CH₃, wherein R′ is selected from the groupconsisting of hydrogen, a C₁₋₂₀ aliphatic group, a monocyclic aromaticgroup as described above, a bicyclic aromatic group as described above,and a multicyclic aromatic group as described above, and R″ is selectedfrom the group consisting of hydrogen, a C₁₋₂₀ aliphatic group, amonocyclic aromatic group as described above, a bicyclic aromatic groupas described above, a multicyclic aromatic group as described above, aC₁₋₂₀ alicyclic group, a noncarbon/nonoxygen moiety as described above,a carbohydrate, a lipid, a nucleic acid and a protein, and m≦30. Any oneof R₆, R₇, R₈ and R₉ can be attached covalently or noncovalently to apolymer of synthetic or natural origin. In Formula I, one of R₆ and R₇and one of R₈ and R₉ can be absent such that a double bond joins the twocarbon atoms to which the remaining R groups are attached. In Formula I,n=0-20, and in Formula II, n=1-20. X is a heteroatom, and R₁₀ and R₁₁are independently selected from the group consisting of a C₁₋₂₀aliphatic group, a monocyclic aromatic group as described above, abicyclic aromatic group as described above, a multicyclic aromatic groupas described above, each as defined above, a C₁₋₂₀ aliphatic/aromaticgroup, a heteroatomic group, a C₁₋₂₀ ether-containing group, a C₁₋₂₀keto group, a C₁₋₂₀ aldehydic group, a carboxamido group, a cyano group,an amino group, a carboxyl group, a selenium-containing group, a sulfatogroup, a sulfito group, a sulfenato group, a sulfinato group, and asulfonato group. R₁₀ and R₁₁ can be connected through an aliphatic groupand/or an aromatic group, or R₁₀ and/or R₁₁ can comprise a memberselected from the group consisting of a carbohydrate, a lipid, a nucleicacid and a protein. Also provided by the present invention is acomposition comprising a nitroxide or a prodrug thereof for use in theabove-described method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of tumor-free survival (%) vs. time (days), whereinopen circles represent the control animals and closed circles representthe nitroxide treated animals.

FIG. 2 is a graph of total number of tumors/group (n=20) versuscontrol-1, control-2, Tempol/1 year, and Tempol/entire life span groups.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for the prophylactic andtherapeutic treatment of cancer in an animal, preferably a mammal, morepreferably a human. The cancer can be due to a genetic defect, such as apoint mutation, an insertion or a deletion, which can be eitherhomozygous or heterozygous, in (i) a tumor suppressor gene, such thatthe tumor suppressor gene no longer suppresses tumor formation or doesso with reduced efficacy, or (ii) a protooncogene, such that theprotooncogene is converted to an oncogene, which causes cancer. Examplesof inherited genetic defects that predispose humans to developing cancerinclude, but are not limited to, ataxia telangiectasia, Cowden'sdisease, Torre's syndrome, Gardner's syndrome, Wiskott-Aldrich syndrome,Peutz-Jeghers syndrome, Bloom's syndrome, Fanconi's syndrome, Wemerssyndrome, Chediak-Higashi syndrome, retinoblastoma, Beckwith-Wiedemansyndrome, and neuroblastoma. In addition to cancers arising from suchinherited genetic defects, genetic defects can be induced by a varietyof agents that damage DNA. For example, a number of studies have shownthat oxidizing agents (e.g., ionizing radiation and/or oxygen derivedfree radicals) increase DNA mutations, leading to cancer induction inmammals (see, e.g., Helbock et al., PNAS USA 95: 288-293 (1998);Kreutzer et al., PNAS USA 95: 3578-3582 (1998); Valentine et al.,Biochemistry 37: 7030-7038 (1998); McBride et al., Biochemistry 30:207-213 (1991); Reid et al., Princess Takamatsu Symp. 22: 221-229(1991); and Klaunig et al., Environ. Health Perspect. 106 (Suppl.):289-95 (1998)).

Genetic “knock-out” models can be developed for genetic defects inaccordance with methods known in the art (Joyner et al., Nature 338:153-156 (1989); see also Donehower et al. (1992), supra, and Harvey etal. (1993), supra) so as to determine whether or not a cancer caused bysuch a defect can be prevented, its onset delayed, and/or itsprogression slowed by a nitroxide or prodrug thereof in accordance withthe present invention. Such models then can be used further to determinewhich nitroxides or prodrugs thereof are particularly effective in theprophylactic and therapeutic treatment of a given cancer, and in whatamounts. A genetic “knock-out” model has been developed for ataxiatelangiectasia (Barlow et al., Cell 86: 159-171 (1996)).

The method of the present invention comprises administering to ananimal, preferably a mammal, more preferably a human, at risk fordeveloping a cancer or having a cancer (e.g., a genetic defect or aproclivity for a genetic defect, such as an induced or inherited geneticdefect, that promotes or causes cancer), a nitroxide or a prodrugthereof in an amount sufficient to prevent or treat said cancer,respectively, wherein said cancer is susceptible to prevention ortreatment with said nitroxide or said prodrug thereof. By “nitroxide” ismeant a compound that contains one or more nitroxide groups (i.e., N—O.groups). By “prodrug” is meant a compound that contains at least onefunctional group that can be converted into a nitroxide group, therebytransforming the prodrug into a nitroxide.

If the cancer is caused by a genetic defect, preferably the geneticdefect affects a cancer regulatory gene or a tumor suppressor gene. Acancer regulatory gene is a gene that up-regulates or down-regulates agene that causes cancer. Examples of such a gene include abl and bcl2. Atumor suppressor gene is a gene that suppresses tumor formation, such asthe p53 gene, which is preferred.

The nitroxide or prodrug thereof to be administered preferably isalicyclic or heterocyclic. More preferably, the alicyclic orheterocyclic nitroxide or prodrug thereof is a compound of Formula I orFormula II:

wherein R₁ is selected from the group consisting of H, OH, OZ, O., ═Oand Y, wherein Y is a leaving group, which can be converted to H, OH, Oor ═O by reaction with a nucleophilic agent, and Z is selected from thegroup consisting of a C₁₋₂₀ aliphatic group, a monocyclic aromaticgroup, a bicyclic aromatic group, a multicyclic aromatic group, a C₁₋₂₀alicyclic group, a noncarbon/nonoxygen moiety, a carbohydrate, a lipid,a nucleic acid and a protein. Preferably, the aromatic group comprises a5- or 6-membered structure in which each member is independentlyselected from the group consisting of carbon and a heteroatom. Preferredheteroatoms in the aromatic group include nitrogen, oxygen, sulfur,phosphorus and boron. The noncarbon/nonoxygen moiety preferablycomprises a member selected from the group consisting of boron, sulfur,phosphorus and nitrogen. R₂, R₃, R₄ and R₅ are independently selectedfrom the group consisting of a C₁₋₂₀ alkyl group, a C₂₋₂₀ alkenyl group,a C₂₋₂₀ alkynyl group, and —CH₂—[CR′R″]_(m)—CH₃, wherein R′ is selectedfrom the group consisting of hydrogen, a C₁₋₂₀ aliphatic group, amonocyclic aromatic group as described above, a bicyclic aromatic groupas described above, and a multicyclic aromatic group as described above,and R″ is selected from the group consisting of hydrogen, a C₁₋₂₀aliphatic group, a monocyclic aromatic group as described above, abicyclic aromatic group as described above, a multicyclic aromatic groupas described above, a C₁₋₂₀ alicyclic group, a noncarbon/nonoxygenmoiety as described above, a carbohydrate, a lipid, a nucleic acid, anda protein, and m≦30. R₂ and R₃ or R₄ and R₅ can be connected through oneor more members, each of which is independently selected from the groupconsisting of carbon and a heteroatom. R₆, R₇, R₈ and R₉ areindependently selected from the group consisting of hydrogen, a hydroxylgroup, a C₁₋₂₀ aldehydic group, a C₁₋₂₀ keto group, a primary aminogroup, a secondary amino group, a tertiary amino group, a sulfido group,a disulfido group, a sulfato group, a sulfito group, a sulfonato group,a sulfinato group, a sulfenato group, a sulfamato group, ametal-containing group, wherein the metal is preferably selected fromthe group consisting of a transition metal and a lanthanide, a siliconegroup, a halide, a C₁₋₂₀ ester-containing group, a carboxyl group, aphosphato group, a phosphino group, a phosphinato group, a phosphonatogroup, a C₁₋₂₀ alkyl group, a C₂₋₂₀ alkenyl group, a C₂₋₂₀ alkynylgroup, and —CH₂—[CR′R″]_(m)—CH₃, wherein R′ is selected from the groupconsisting of hydrogen, a C₁₋₂₀ aliphatic group, a monocyclic aromaticgroup as described above, a bicyclic aromatic group as described above,and a multicyclic aromatic group as described above, and R″ is selectedfrom the group consisting of hydrogen, a C₁₋₂₀ aliphatic group, amonocyclic aromatic group as described above, a bicyclic aromatic groupas described above, a multicyclic aromatic group as described above, aC₁₋₂₀ alicyclic group, a noncarbon/nonoxygen moiety as described above,a carbohydrate, a lipid, a nucleic acid and a protein, and m≦30. Any oneof R₆, R₇, R₈ and R₉ can be attached covalently or noncovalently to apolymer of synthetic or natural origin. In Formula I, one of R₆ and R₇and one of R₈ and R₉ can be absent such that a double bond joins the twocarbon atoms to which the remaining R groups are attached. In Formula I,n=0-20, and in Formula II, n=1-20. X is a heteroatom, and R₁₀ and R₁₁are independently selected from the group consisting of a C₁₋₂₀aliphatic group, a monocyclic aromatic group as described above, abicyclic aromatic group as described above, a multicyclic aromatic groupas described above, a C₁₋₂₀ aliphatic/aromatic group, a heteroatomicgroup, a C₁₋₂₀ ether-containing group, a C₁₋₂₀ keto group, a C₁₋₂₀aldehydic group, a carboxamido group, a cyano group, an amino group, acarboxyl group, a selenium-containing group, a sulfato group, a sulfitogroup, a sulfenato group, a sulfinato group, and a sulfonato group. R₁₀and R₁₁ can be connected through an aliphatic group and/or an aromaticgroup, or R₁₀ and/or R₁₁ can comprise a member selected from the groupconsisting of a carbohydrate, a lipid, a nucleic acid and a protein. Thealiphatic group can be branched, substituted and/or unsaturated. If thealiphatic group is substituted, preferably it is substituted with aheteroatom, which is preferably selected from the group consisting ofoxygen, phosphorus, selenium, sulfur and nitrogen. The aromatic groupcan be substituted. If the aromatic group is substituted, preferably itis substituted with a heteroatom, which is preferably selected from thegroup consisting of nitrogen, oxygen, sulfur, phosphorus and boron. Thealicyclic group can be substituted and/or unsaturated. If the alicyclicgroup is substituted, preferably it is substituted with a heteroatom.The amino group also can be substituted. If the amino group issubstituted, preferably it is substituted with up to three substituentsselected from the group consisting of a C₁₋₂₀ aliphatic group, amonocyclic aromatic group, a bicyclic aromatic group, a multicyclicaromatic group, and a C₁₋₂₀ alicyclic group, all of which are asdescribed above. Although carbon ranges have been specified for a numberof the substituents recited above, such carbon ranges are onlypreferred, as substituents comprising carbon atoms outside the specifiedranges can be effective in the context of the present inventive method.

The above-described method can be adapted for in vitro utilization forscientific and research purposes, including the determination of whichtypes of cancers can be treated by administration of a nitroxide or aprodrug thereof in accordance with the present inventive method.However, the above-described method has particular usefulness in in vivoapplications, e.g., in the prevention, delay of onset, and/or slowing ofthe progression of cancer.

One skilled in the art will appreciate that many suitable methods ofadministering a nitroxide or a prodrug thereof to an animal, preferablya mammal, more preferably a human, are available, that more than oneroute can be used to administer a particular compound, and that aparticular route can provide a more immediate and more effectivetreatment than another route. Accordingly, the above-described method ismerely exemplary and is in no way limiting.

The dose administered to an animal, preferably a mammal, more preferablya human, with an induced and/or inherited genetic defect that causes orpromotes cancer, should be sufficient to prevent cancer, delay itsonset, and/or slow its progression. One skilled in the art willrecognize that the dosage will depend upon a variety of factors,including the potency of the particular compound employed, and the age,species, condition, and body weight of the animal. The size of the dosewill also be determined by the route, timing and frequency ofadministration, as well as the existence, nature, and extent of anyadverse side-effects that might accompany the administration of aparticular compound, and the desired physiological effect.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages, which are lessthan the optimal dose of the compound. Thereafter, the dosage isincreased by increments until the optimal effect under the circumstancesis reached. The present inventive method will typically involve theadministration of about 0.1 to about 100 mg of one or more of thecompounds described above per kg of body weight.

The present invention also provides a composition comprising a nitroxideor prodrug thereof, preferably an alicyclic or heterocyclic nitroxide orprodrug thereof, more preferably a compound of Formula I or Formula II,as described above. Compounds of Formula I or II can be synthesizedaccording to methods that are well known in the art. See, for example,Rosantzev, “Synthesis of Individual Radicals,” Chapter III, pp. 67-89,and “Synthesis of Some Stable Radicals and the Most ImportantIntermediates,” Chapter IX, pp. 203-247, In Free Nitroxyl Radicals,Plenum Press (1970). Preferably, the composition is a pharmaceuticalcomposition, which comprises a pharmaceutically acceptable carrier. Anysuitably carrier can be used, and will typically be chosen uponconsideration of its chemico-physical properties, such as solubility anddegree of reactivity with the other components of the composition, andby the route of administration. It will be appreciated by one of skillin the art that, in addition to the following described pharmaceuticalcomposition, the compounds of the present inventive method can beformulated as inclusion complexes, such as cyclodextrin inclusioncomplexes, or liposomes, for example.

Examples of pharmaceutically acceptable acid addition salts for use inthe present inventive pharmaceutical composition include those derivedfrom mineral acids, such as hydrochloric, hydrobromic, phosphoric,metaphosphoric, nitric and sulfuric acids, and organic acids, such astartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic,gluconic, succinic, and arylsulfonic, for example p-toluenesulfonicacids.

The pharmaceutically acceptable excipients described herein, forexample, vehicles, adjuvants, carriers or diluents, are well-known tothose who are skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone which is chemically inert to the active compounds and one which hasno detrimental side effects or toxicity under the conditions of use.

The choice of excipient will be determined in part by the particularcompound, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of the pharmaceutical composition of the present invention.The following formulations for oral, aerosol, parenteral, subcutaneous,intravenous, intramuscular, interperitoneal, rectal, and vaginaladministration are merely exemplary and are in no way limiting.Injectable formulations are among those formulations that are preferredin accordance with the present inventive methods. The requirements foreffective pharmaceutical carriers for injectable compositions are wellknown to those of ordinary skill in the art (See Pharmaceutics andPharmacy Practice, J.B. Lippincott Company, Philadelphia, Pa., Bankerand Chalmers, eds., pages 238-250, (1982), and ASHP Handbook onInjectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). It ispreferred that such injectable compositions be administeredintravenously, intratumorally (within the tumor), or peritumorally (nearthe outside of the tumor). It will be appreciated by one of skill in theart that various of the described injectable compositions are suitablefor intratumoral and peritumoral administration.

Topical formulations are well-known to those of skill in the art. Suchformulations are suitable in the context of the present invention forapplication to skin.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the nitroxide or prodrugthereof dissolved in diluents, such as water, saline, or orange juice;(b) capsules, sachets, tablets, lozenges, and troches, each containing apredetermined amount of the nitroxide or prodrug thereof, as solids orgranules; (c) powders; (d) suspensions in an appropriate liquid; and (e)suitable emulsions. Liquid formulations may include diluents, such aswater and alcohols, for example, ethanol, benzyl alcohol, and thepolyethylene alcohols, either with or without the addition of apharmaceutically acceptable surfactant, suspending agent, or emulsifyingagent. Capsule forms can be of the ordinary hard- or soft-shelledgelatin type containing, for example, surfactants, lubricants, and inertfillers, such as lactose, sucrose, calcium phosphate, and corn starch.Tablet forms can include one or more of lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,and other excipients, colorants, diluents, buffering agents,disintegrating agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible excipients. Lozenge forms cancomprise the nitroxide or prodrug thereof, flavoring, for example,sucrose and acacia or tragacanth, as well as pastilles comprising thenitroxide or prodrug thereof in an inert base, such as gelatin andglycerin, or sucrose and acacia, emulsions, gels, and the likecontaining, in addition to the nitroxide or prodrug thereof, suchexcipients as are known in the art.

The nitroxides and prodrugs thereof, alone or in combination with othersuitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer. Such spray formulations also may be usedto spray mucosa.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The nitroxide or prodrug thereof can be administered in aphysiologically acceptable diluent in a pharmaceutical carrier, such asa sterile liquid or mixture of liquids, including water, saline, aqueousdextrose and related sugar solutions, alcohols, such as ethanol,isopropanol, and hexadecyl alcohol, glycols, such as propylene glycoland polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane, ethers, such aspoly(ethylene glycol) 400, oils, fatty acids, fatty acid esters orglycerides, and acetylated fatty acid glycerides with or without theaddition of one or more pharmaceutically acceptable surfactants, such assoaps and detergents, suspending agents, such as pectin, carbomers,cellulose derivatives, such as methylcellulose,hydroxypropylmethylcellulose, and carboxymethylcellulose, emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-b-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations will typically contain from about 0.5 toabout 25% by weight of the nitroxide or prodrug thereof in solution.Preservatives and buffers may be used. In order to minimize or eliminateirritation at the site of injection, such compositions may contain oneor more nonionic surfactants having a hydrophile-lipophile balance (HLB)of from about 12 to about 17. The quantity of surfactant in suchformulations will typically range from about 5 to about 15% by weight.Suitable surfactants include polyethylene sorbitan fatty acid esters,such as sorbitan monooleate and the high molecular weight adducts ofethylene oxide with a hydrophobic base, formed by the condensation ofpropylene oxide with propylene glycol. The parenteral formulations canbe presented in unit-dose or multi-dose sealed containers, such asampoules and vials, and can be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid excipient,for example, water, for injections, immediately prior to use.Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.

Additionally, the nitroxides and prodrugs thereof can be made intosuppositories by mixing with a variety of bases, such as emulsifyingbases or water-soluble bases. Formulations suitable for vaginaladministration may be presented as pessaries, tampons, creams, gels,pastes, foams, or spray formulas containing, in addition to thenitroxide or prodrug thereof, such carriers as are known in the art tobe appropriate.

EXAMPLES

The following examples further illustrate the present invention and, ofcourse, should not be construed as in any way limiting its scope.

Example 1

This example demonstrates that administration of a nitroxide to p53−/−mice delays the onset of tumors.

Male and female p53−/− mice (strain 129/Sv-Trp5n^(tml Tyi)) werepurchased from Jackson Labs (Bar Harbor, Me.). Such animals uniformlydie within a few months after birth due to rapid tumor formation andgrowth. Animals arrived in the laboratory at 7-8 weeks of age, wereacclimated for five days and were randomly divided into control (n=8;average weight=24.6 g) and treatment (n=9; average weight=25.0 g)groups. Both groups were allowed food and water ad libitum. The water ofthe control group was supplemented with sugar (4 g/100 ml), whereas thewater of the treatment group was supplemented with sugar (4 g/100 ml)and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) to a finalconcentration of 58 mM. Mice were sacrificed at the first sign of avisible tumor nodule, gross enlargement of the spleen or markeddifficulty in breathing. The results are shown in FIG. 1, which is agraph of tumor-free survival versus time (days), in which closed circlesrepresent the control group and open circles represent the treatedgroup. Daily administration of Tempol to p53−/− mice extended their lifespan by approximately 48% as compared to the control group. TheTempol-treated animals ultimately developed tumors, but the onset oftumor formation was delayed as compared to the control group.

Example 2

This example demonstrates that administration of a nitroxide to normalC3H female mice for their entire life-span decreases the incidence ofcancer in such mice.

Female C3H mice were supplied through the Frederick Cancer ResearchCenter-Animal Production, Frederick, Md. Animals were received at 6weeks of age and were randomly divided into groups (n=20/group) asfollows: Control-1, which received regular food and water; Control-2,which received regular food and water supplemented with sugar (4 g/100ml); Tempol/I Year, which received regular food and water supplementedwith sugar (4 g/100 ml) and Tempol to a final concentration of 58 mM forone year, after which they were converted to regular food and water; andTempol/Entire Life Span, which received regular food and watersupplemented with sugar (4 g/100 ml) and Tempol to a final concentrationof 58 mM for their entire life span. All groups were allowed food andwater ad libitum. All groups were followed for their entire life span.Animals were sacrificed at the first sign of a visible tumor nodule,gross enlargement of spleen, or marked difficulty in breathing. Thepresence of tumor was confirmed histologically.

The results are shown in FIG. 2, which is a graph of the total number oftumors versus the various groups. Administration of Tempol in thedrinking water for one year dramatically reduced the incidence of cancerin the treated animals compared to both control groups, andadministration of Tempol present in the drinking water for the entirelife span of the animals further reduced the incidence of cancer(four-fold reduction compared to controls). Nitroxide treatmenteffectively reduced the incidence of cancer.

All publications cited herein are hereby incorporated by reference tothe same extent as if each publication were individually andspecifically indicated to be incorporated by reference and were setforth in its entirety herein.

While this invention has been described with emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat the preferred embodiments may be varied. It is intended that theinvention may be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the appended claims.

1. A method for the prophylactic or therapeutic treatment of cancer inan animal, which method comprises administering to an animal at risk fordeveloping a cancer or having a cancer a nitroxide or a prodrug thereofin an amount sufficient to prevent or treat said cancer, wherein saidcancer is susceptible to prevention or treatment by said nitroxide orprodrug thereof.
 2. The method of claim 1, wherein said nitroxide orprodrug thereof is alicyclic or heterocyclic.
 3. The method of claim 2,wherein said nitroxide or prodrug thereof is a compound of Formula I orII:

wherein R₁ is selected from the group consisting of H, OH, OZ, O., ═Oand Y, wherein Y is a leaving group, which can be converted to H, OH, Oor ═O by reaction with a nucleophilic agent, and Z is selected from thegroup consisting of a C I₂₀ aliphatic group, a monocyclic aromaticgroup, a bicyclic aromatic group, a multicyclic aromatic group, a C₁₋₂₀alicyclic group, a noncarbon/nonoxygen moiety, a carbohydrate, a lipid,a nucleic acid and a protein, wherein R₂, R₃, R₄ and R₅ areindependently selected from the group consisting of a C₁₋₂₀ alkyl group,a C₂₋₂₀ alkenyl group, a C₂₋₂₀ alkynyl group, and —CH₂—[CR′R″]_(m)—CH₃,wherein R′ is selected from the group consisting of hydrogen, a C₁₋₂₀aliphatic group, a monocyclic aromatic group, a bicyclic aromatic group,and a multicyclic aromatic group, and R″ is selected from the groupconsisting of hydrogen, a C₁₋₂₀ aliphatic group, a monocyclic aromaticgroup, a bicyclic aromatic group, a multicyclic aromatic group, a C₁₋₂₀alicyclic group, a noncarbon/nonoxygen moiety, a carbohydrate, a lipid,a nucleic acid, and a protein, m≦30, and R₂ and R₃ or R₄ and R₅ can beconnected through one or more members, each of which is independentlyselected from the group consisting of carbon and a heteroatom, whereinR₆, R₇, R₈ and R₉ are independently selected from the group consistingof hydrogen, a hydroxyl group, a C₁₋₂₀ aldehydic group, a C₁₋₂₀ ketogroup, a primary amino group, a secondary amino group, a tertiary aminogroup, a sulfido group, a disulfido group, a sulfato group, a sulfitogroup, a sulfonato group, a sulfinato group, a sulfenato group, asulfamato group, a metal-containing group, a silicone group, a halide, aC₁₋₂₀ ester-containing group, a carboxyl group, a phosphato group, aphosphino group, a phosphinato group, a phosphonato group, a C₁₋₂₀ alkylgroup, a C₂₋₂₀ alkenyl group, a C₂₋₂₀ alkynyl group, and—CH₂—[CR′R″]_(m)—CH₃, wherein R′ is selected from the group consistingof hydrogen, a C₁₋₂₀ aliphatic group, a monocyclic aromatic group, abicyclic aromatic group, and a multicyclic aromatic group, and R″ isselected from the group consisting of hydrogen, a C₁₋₂₀ aliphatic group,a monocyclic aromatic group, a bicyclic aromatic group, a multicyclicaromatic group, a C₁₋₂₀ alicyclic group, a noncarbon/nonoxygen moiety, acarbohydrate, a lipid, a nucleic acid and a protein, and m≦30, andwherein any one of R₆, R₇, R₈ and R₉ can be attached covalently ornoncovalently to a polymer of synthetic or natural origin, wherein inFormula I, one of R₆ and R₇ and one of R₈ and R₉ can be absent such thata double bond joins the two carbon atoms to which the remaining R groupsare attached, wherein n=0-20 in Formula I, and n=1-20 in Formula II,wherein X is a heteroatom, and wherein R₁₀ and R₁₁ are independentlyselected from the group consisting of a C₁₋₂₀ aliphatic group, amonocyclic aromatic group, a bicyclic aromatic group, a multicyclicaromatic group, a C₁₋₂₀ aliphatic/aromatic group, a heteroatomic group,a C₁₋₂₀ ether-containing group, a C₁₋₂₀ keto group, a C₁₋₂₀ aldehydicgroup, a carboxamido group, a cyano group, an amino group, a carboxylgroup, a selenium-containing group, a sulfato group, a sulfito group, asulfenato group, a sulfinato group, and a sulfonato group, and whereinR₁₀ and R₁₁ can be connected through an aliphatic group and/or anaromatic group, or R₁₀ and/or R₁₁ can comprise a member selected fromthe group consisting of a carbohydrate, a lipid, a nucleic acid and aprotein.
 4. The method of claim 3, wherein said aliphatic group isbranched, substituted and/or unsaturated.
 5. The method of claim 4,wherein said aliphatic group is substituted with a member selected fromthe group consisting of oxygen, phosphorus, selenium, sulfur andnitrogen.
 6. The method of claim 3, wherein said aromatic groupcomprises a five- or six-membered ring, in which each of the five or sixmembers is independently selected from the group consisting of carbonand a heteroatom.
 7. The method of claim 6, wherein said heteroatom isselected from the group consisting of nitrogen, oxygen, sulfur,phosphorus and boron.
 8. The method of claim 3, wherein the metal ofsaid metal-containing group is selected from the group consisting of atransition metal and a lanthanide.
 9. The method of claim 6, whereinsaid aromatic group is substituted.
 10. The method of claim 9, whereinsaid aromatic group is substituted with a heteroatom.
 11. The method ofclaim 10, wherein said heteroatom is selected from the group consistingof nitrogen, oxygen, sulfur, phosphorus and boron.
 12. The method ofclaim 3, wherein said alicyclic group is substituted and/or unsaturated.13. The method of claim 11, wherein said alicyclic group is substitutedwith a heteroatom.
 14. The method of claim 3, wherein said amino groupis substituted.
 15. The method of claim 14, wherein said amino group issubstituted with up to three substituents selected from the groupconsisting of a C₁₋₂₀ aliphatic group, a monocyclic aromatic group, abicyclic aromatic group, a multicyclic aromatic group, and a C₁₋₂₀alicyclic group.
 16. The method of claim 15, wherein said aromatic groupcomprises a five- or six-membered ring, in which each of the five or sixmembers is independently selected from the group consisting of carbonand a heteroatom.
 17. The method of claim 16, wherein said heteroatom isselected from the group consisting of nitrogen, oxygen, sulfur,phosphorus and boron.
 18. The method of claim 15, wherein said aromaticgroup is substituted.
 19. The method of claim 18, wherein said aromaticgroup is substituted with a heteroatom.
 20. The method of claim 19,wherein said heteroatom is selected from the group consisting ofnitrogen, oxygen, sulfur, phosphorus and boron.
 21. The method of claim3, wherein said noncarbon/nonoxygen moiety is selected from the groupconsisting of boron, sulfur, nitrogen and phosphorus.
 22. The method ofclaim 1, wherein said cancer is due to a genetic defect of a cancerregulatory gene or a tumor suppressor gene.
 23. The method of claim 22,wherein said tumor suppressor gene is the p53 gene.
 24. The method ofclaim 2, wherein said cancer is due to a genetic defect of a cancerregulatory gene or a tumor suppressor gene.
 25. The method of claim 24,wherein said tumor suppressor gene is the p53 gene.
 26. The method ofclaim 3, wherein said cancer is due to a genetic defect of a cancerregulatory gene or a tumor suppressor gene.
 27. The method of claim 26,wherein said tumor suppressor gene is the p53 gene.